Organisms regulate expression of their genome by means of a diverse repertoire of molecular mechanisms and circuitry. Yet, little is known about the forces that lead to the selection or maintenance of a specific mechanism for the regulation of a given set of genes in a particular organism. Is this process random, or is it governed by rules? The answer to this basic question is important for understanding the evolution of gene regulation. It also is important for redirecting normal expression for biotechnological purposes or correcting pathological expression for therapeutic purposes. The long-term objective of this project is to relate function, design, and evolution of integrated biological systems to their underlying molecular determinants. This will require analysis of the systemic function of particular gene circuits and biochemical mechanisms, classification of these into specific categories of design, and examination of their natural selection and maintenance. The specific aims are to elucidate (1) the designs for coupling elementary gene circuits in repressible systems, (2) the factors that influence the evolution of alternative molecular modes of gene control, (3) the functional significance of different strategies for biochemical signal transduction, and (4) the integrated behavior of a system of elementary gene circuits under the hierarchical control of a global regulator. The methodology emphasizes mathematical and computer-assisted analyses because of their unique ability to systematically relate integrated function and design of complex systems to their molecular elements. The general outline for the analysis in each case is as follows: Specific models based on known or suspected molecular elements and interactions are formulated; their integrated behavior is analyzed and compared according to several criteria for functional effectiveness; the results are interpreted in terms of optimal designs for specific functions; and, finally, the biological significance of the results is addressed and predictions are made for experimental testing. This approach is helping to create a more rational basis for predicting the integrated behavior of complex biological systems. Accurate predictions of this nature will be necessary if we are to understand more fully the normal systemic processes of homeostasis, growth and development, or their pathological manifestations such as metabolic diseases, developmental abnormalities and cancer. A focus on prokaryotic organisms is important not only because they serve as model systems for understanding higher organisms, but also because of their role in the pathogenesis of infectious diseases.